Over the past 3 decades, the incidence of esophageal adenocarcinoma (EAC) has risen faster than any other cancer in developed countries, a trend that is expected to continue with increasing prevalence of obesity and acid reflux. This disease is believed to arise from Barrett's esophagus, where early cancer detection is currently performed on white light endoscopy with random biopsies. This approach is prone to sampling error because high-grade dysplasia (HGD) and early EAC are difficult to detect on imaging because of flat morphology, patchy distribution, and low prevalence. The genes for epithelial growth factor receptors, EGFR and ErbB2, are amplified in high frequency in EAC with minimal overlap among individual patients. The corresponding protein targets are expressed on the cell surface and accessible to in vivo imaging with use of highly specific peptides. We will develop a multimer that links individual peptides specific for either EGFR or ErbB2, and attach Cy5.5, a bright near-infrared (NIR) fluorophore for imaging. We will optimize the peptides on alignment to these targets using a structural model, and validate specific binding on competition and siRNA knockdown studies. We will characterize binding affinity and rapid binding on flow cytometry, and validate binding of the optimized multimer on human specimens of esophageal neoplasia ex vivo using a multi-modal NIR endoscope. Fluorescence and reflectance images will be ratioed to correct for difference in distance and geometry over the image field-of-view. The threshold that maximizes variance will be used to create a binary image. The contour will define a red-flag region on the white light image to help physicians guide tissue resection. These experiments will verify instrument sensitivity to target overexpression. We will then perform a pharmacology toxicology study in animals to establish multimer safety, and submit an Investigational New Drug Application (IND) to the FDA to perform a first-in-human clinical study. The multimer will first be evaluated in patients to establish safety and then studied on imaging to validate binding to early neoplasia. The results will be compared with histology. Successful completion of these aims will provide the medical community with an integrated multi modal imaging methodology that detects overexpressed cell surface targets to guide tissue resection of lesions that have increased risk for progressing onto cancer, detect cancer at the earliest stages possible, reduce overdiagnosis, and distinguish lethal from non-lethal disease. This collaborative work will be performed by a multi-disciplinary team at the University of Michigan, including TD Wang who will develop the peptide multimer, DG Beer who will validate specific multimer binding to EGFR and ErbB2, and DK Turgeon who will perform the clinical studies. This experienced team has recently completed an FDA-approved first-in-human imaging study of a monomer peptide in esophagus.